Recent trends toward the production of “green” chemicals will require development of innovative synthesis techniques that are highly efficient and cost effective.
Throughout the past decade, a number of traditional chemical companies in the United States and Europe have begun to develop infrastructures for the production of compounds using biocatalytic processes. Considerable progress has been reported toward new processes for commodity chemicals such as ethanol (Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999 “Enteric bacterial catalyst for fuel ethanol production” Biotechnol. Prog. 15:855-866; Underwood, S. A., S. Zhou, T. B. Causey, L. P. Yomano, K. T. Shanmugam, and L. O. Ingram, 2002 “Genetic changes to optimize carbon partitioning between ethanol and biosynthesis in ethanologenic Escherichia coli.” Appl. Environ. Microbiol. 68:6263-6272), lactic acid (Zhou, S., T. B. Causey, A. Hasona, K. T. Shanmugam and L. O. Ingram, 2003 “Production of optically pure D-lactic acid in mineral salts medium by metabolically engineered Escherichia coli W3110” Appl. Environ. Microbiol. 69:399-407; Chang, D., S. Shin, J. Rhee, and J. Pan, 1999 “Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1” Appl. Environ. Microbiol. 65:1384-1389; Dien, B. S., N. N. Nichols, and R. J. Bothast, 2001 “Recombinant Escherichia coli engineered for the production of L-lactic acid from hexose and pentose sugars” J. Ind Microbiol. Biotechnol. 27:259-264), 1,3-propanediol (Nakamura, U.S. Pat. No. 6,013,494; Tong, I., H. H. Liao, and D. C. Cameron, 1991 “1,3-propanediol production by Escherichia coli expressing genes from the Klebsiella-pneumoniae-DHA regulon” App. Env. Microbiol. 57:3541-3546), and adipic acid (Niu, W., K. M. Draths, and J. W. Frost, 2002 “Benzene-free synthesis of adipic acid” Biotechnol. Prog. 18:201-211).
In addition, advances have been made in the genetic engineering of microbes for higher value specialty compounds such as acetate, polyketides (Beck, B. J., C. C. Aldrich, R. A. Fecik, K. A. Reynolds, and D. H. Sherman, 2003 “Iterative chain elongation by a pikromycin monomodular polyketide synthase” J. Am. Chem. Soc. 125:4682-4683; Dayem, L. C., J. R. Carney, D. V. Santi, B. A. Pfeifer, C. Khosla, and J. T. Kealey, 2002 “Metabolic engineering of a methylmalonyl-CoA mutase—epimerase pathway for complex polyketide biosynthesis in Escherichia coli.” Biochem. 41:5193-5201) and carotenoids (Wang, Chia-wei, Min-Kyu Oh, J. C. Liao, 2000 “Directed evolution of metabolically engineered Escherichia coli for carotenoid production” Biotechnol. Prog. 16:922-926).
Acetic acid, a widely used specialty chemical in the food industry, has recently emerged as a potential bulk chemical for the production of plastics and solvents. Acetic acid has been produced using microbial systems; however, the production of acetic acid in microbial systems competes with the production of CO2 and cell mass. Thus, while efficient acetate-producing microbial systems are important for industrial uses, the systems must have an increased output of acetate with a decreased input of expensive microbial nutrients.
The biological production of acetic acid has been largely displaced by petrochemical routes as the uses for this commodity chemical have expanded from food products to plastics, solvents, and road de-icers (Freer, S. N., 2002 “Acetic acid production by Dekkera/Brettanomyces yeasts” World J. Microbiol. Biotechnol. 18:271-275). In 2001, the world production of acetic acid reached an estimated 6.8 million metric tons, half of which was produced in the United States.
Previously, three microbial approaches have been explored for acetic acid production. In the two-step commercial process, sugars are fermented to ethanol by Saccharomyces yeast. Then, the resulting beers are oxidized to acetic acid by Acetobacter under aerobic conditions (Berraud, C., 2000 “Production of highly concentrated vinegar in fed-batch culture” Biotechnol. Lett. 22:451-454; Cheryan, M., S. Parekh, M. Shah, and K. Witjitra, 1997 “Production of acetic acid by Clostridium thermoaceticum” Adv. Appl. Microbial. 43:1-33). Using this process, acetic acid titres of around 650 mM are typically produced; however, higher titres can be readily achieved by the addition of complex nutrients in fed-batch processes requiring 60-120 hours. Overall yields for this commercial process have been estimated to be 76% of the theoretical maximum (2 acetate per glucose; 0.67 g acetic acid per g glucose).
Under a second approach, carbohydrates can be anaerobically metabolized to acetic acid at substantially higher yields (3 acetates per glucose) by Clostridia that contain the Ljungdahl-Wood pathway for acetogenesis (Berraud, C., 2000 “Production of highly concentrated vinegar in fed-batch culture” Biotechnol. Lett. 22:451-454; Ljungdahl, L. G., 1986 “The autotrophic pathway of acetate synthesis in acetogenic bacteria” Ann. Rev. Microbiol. 40:415-450). In particular, Clostridium thermoaceticum containing the Ljungdahl-Wood pathway produces high yields of acetic acid (Cheryan, M., S. Parekh, M. Shah, and K. Witjitra, 1997 “Production of acetic acid by Clostridium thermoaceticum” Adv. Appl. Microbial. 43:1-33).
Recently, Freer (Freer, S. N., 2002 “Acetic acid production by Dekkera/Brettanomyces yeasts” World J. Microbial. Biotechnol. 18:271-275) identified yeast strains (Dekkera and Brettanomyces) that produce acetic acid as a primary product from glucose for potential use in acetic acid production. All three of these current microbial acetic acid production systems require complex nutrients, which increase the cost of materials, acetate purification, and waste disposal.
Escherichia coli is widely used as a biocatalyst for high value products such as recombinant proteins (Akesson, M., P. Hagander, and J. P. Axelsson, 2001 “Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding” Biotechnol. Bioeng. 73:223-230; Aristidou, A. A., K. San, and G. N. Bennett, 1995 “Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction” Biotechnol. Prog. 11:475-478; Contiero, J., C. Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe, 2000 “Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli” J. Ind. Microbiol. 24:421-430; Luli, G. W. and R. Strohl, 1990 “Comparison of growth, acetate production and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations” Appl. Environ. Microbiol. 56:1004-1011) and amino acids (Chotani, G., T. Dodge, A. Hsu, M. Kumar, R. LaDuca, D. Trimbur, W. Weyler, and K. Sanford, 2000 “The commercial production of chemicals using pathway engineering” Biochem. Biophys. Acta 1543:434-455; Eggeling, L., W. Pfefferle, and H. Sahm, 2001 “Amino acids,” p. 281-304 in C. Ratledge and B. Kristiansen (ed.), Basic Biotechnology, 2″ edition. Cambridge University Press. Cambridge, U.K.).
Escherichia coli generate acetyl˜CoA during fermentative and oxidative metabolism, which the cell then uses to produce small amounts of acetate (Akesson, M., P. Hagander, and J. P. Axelsson, 2001 “Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding” Biotechnol. Bioeng. 73:223-230; Contiero, J., C. Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe, 2000 “Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli” J. Ind. Microbiol. 24:421-430).
Many E. coli strains grow well in simple mineral salts medium and readily metabolize all of the hexose and pentose sugar constituents of plant biomass (Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999 “Enteric bacterial catalyst for fuel ethanol production” Biotechnol. Prog. 15:855-866). During aerobic and anaerobic carbohydrate metabolism, acetate is typically produced as a minor product. Recent successes have been reported in the engineering of E. coli strains for commodity chemicals such as propanediol (Nakamura, C. E., A. A. Gatenby, Hsu, A. K.-H., R. D. LaReau, S. L. Haynie, M. Diaz-Torres, D. E. Trimbur, G. M. Whited, V. Nagarajan, M. S. Payne, S. K. Picataggio, and R. V. Nair, 2000 “Method for the production of 1,3-propanediol by recombinant microorganisms” U.S. Pat. No. 6,013,494; Tong, I., H. H. Liao, and D. C. Cameron, 1991 “1,3-propanediol production by Escherichia coli expressing genes from the Klebsiella-pneumoniae-DHA regulon” App. Env. Microbiol. 57:3541-3546), adipic acid (Niu, W., K. M. Draths, and J. W. Frost, 2002 “Benzene-free synthesis of adipic acid” Biotechnol. Prog. 18:201-211), lactic acid (Chang, D., S. Shin, J. Rhee, and J. Pan, 1999 “Homofermentative production of D- or L-lactate in metabolically engineered Escherichia coli RR1” Appl. Environ. Microbiol. 65:1384-1389; Dien, B. S., N. N. Nichols, and R. J. Bothast, 2001 “Recombinant Escherichia coli engineered for the production of L-lactic acid from hexose and pentose sugars” J. Ind Microbiol. Biotechnol. 27:259-264), succinic acid (Donnelly, M. I., C. Sanville-Millard, and R. Chatterjee, 1998 “Method for construction of bacterial strains with increased succinic acid production” U.S. Pat. No. 6,159,738; Vemuri, G. N., M. A. Altman, and E. Altman, 2002 “Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli” J. Bacteria 68:1715-1727), and ethanol (Ingram, L. O., H. C. Aldrich, A. C. C. Borges, T. B. Causey, A. Martinez, F. Morales, A. Saleh, S. A. Underwood, L. P. Yomano, S. W. York, J. Zaldivar, and S. Zhou, 1999 “Enteric bacterial catalyst for fuel ethanol production” Biotechnol. Prog. 15:855-866). In using these aerobic and anaerobic processes, the resultant production of acetate by the native pathway (phosphotransacetylase and acetate kinase) has generally been regarded as an undesirable consequence of excessive glycolytic flux (Akesson, M., P. Hagander, and J. P. Axelsson, 2001 “Avoiding acetate accumulation in Escherichia coli cultures using feedback control of glucose feeding” Biotechnol. Bioeng. 73:223-230; Aristidou, A. A., K. San, and G. N. Bennett, 1995 “Metabolic engineering of Escherichia coli to enhance recombinant protein production through acetate reduction” Biotechnol. Prog. 11:475-478; Contiero, J., C. Beatty, S. Kumar, C. L. DeSanti, W. R. Strohl, and A. Wolfe, 2000 “Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli” J. Ind, Microbiol. 24:421-430; Farmer, W. R., and J. C. Liao, 1997 “Reduction of aerobic acetate production by Escherichia coli 1997” Appl. Environ. Microbiol. 63:3205-3210).
Chao and Liao (Chao, Y., and J. C. Liao, 1994 “Metabolic responses to substrate futile cycling in Escherichia coli” J. Biol. Chem. 269:5122-5126) and Patnaik et al. (Patnaik, R., W. D. Roof, R. F. Young, and J. C. Liao, 1992 “Stimulation of glucose catabolism in Escherichia coli by a potential futile cycle” J. Bacteriol. 174:7525-7532) demonstrated a 2-fold stimulation of glycolytic flux in E. coli using plasmids to express genes that created futile cycles to consume ATP.
Recently, Koebmann et al. (Koebmann, B. J., H. V. Westerhoff, J. L. Snoep, D. Nilsson, and P. R. Jensen, 2002 “The glycolytic flux in Escherichia coli is controlled by the demand for ATP” J. Bacteriol. 184:3909-3916) independently concluded that glycolytic flux is limited by ATP utilization during the oxidative metabolism of glucose. In their studies, flux increased in a dose-dependent manner with controlled expression of F1 ATPase from a plasmid. Thus glycolytic flux appears to be regulated by the economy of supply and demand as proposed by Hofmeyer and Cornish-Bowden (Hofmeyer, J.-H. S., and A. Cornish-Bowden, 2000 “Regulating the cellular economy of supply and demand” FEBS Lett. 467:47-51).
Currently, only the two-part commercial process, the Ljungdahl-Wood pathway-containing Clostridia, as well as special yeast strains have been investigated as potential biocatalysts for the production of acetate. Due to the competing production of dicarboxylic acids and cell mass from glucose, the level of acetate production using these methods has been relatively low. Indeed, none of these methods have been reported to grow and produce acetate efficiently in mineral salts media containing sugar. In fact, each of these methods requires the use of complex nutrients, which ultimately increases the cost of materials, acetate purification, and waste disposal. Therefore, a need remains for better biocatalysts that efficiently produce acetate and other fermentation products using a mineral salts medium.
Pyruvic acid is currently manufactured for use as a food additive, nutriceutical, and weight control supplement (Li, Y., J. Chen, and S.-Y. Lun, 2001 “Biotechnological production of pyruvic acid” Appl. Microbiol, Biotechnol. 57:451-459). Pyruvic acid can also be used as a starting material for the synthesis of amino acids such as alanine, tyrosine, phenylalanine, and tryptophan and for acetaldehyde production.
Pyruvate is produced commercially by both chemical and microbial processes. Chemical synthesis involves the decarboxylation and dehydration of calcium tartrate, a by-product of the wine industry. This process involves toxic solvents and is energy intensive with an estimated production cost of $8,650 per ton of pyruvate. Microbial pyruvate production is based primarily on two microorganisms, a multi-vitamin auxotroph of the yeast Torulopsis glabrata (Li, Y., J. Chen, and S.-Y. Lun, and X. S. Rui, 2001 “Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels” Appl. Microbiol. Biotechnol. 55:680-68) and a lipoic acid auxotroph of Escherichia coli containing a mutation in the F1 ATPase component of (F1F0)H+-ATP synthase (Yokota, A., Y. Terasawa, N. Takaoka, H. Shimizu, and F. Tomita, 1994 “Pyruvic acid production by an F1-ATPase-defective mutant of Escherichia coli W1485lip2” Biosci. Biotech, Biochem. 58:2164-2167). Both of these production strains require precise regulation of media composition during fermentation and complex supplements. The estimated production costs of pyruvate production by microbial fermentation with these strains is estimated to be 14.5% ($1,255 per ton pyruvate) of that for chemical synthesis.
Recently, Tomar et al. (Tomar, A., M. A. Eiteman, and E. Altman, 2003 “The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli.” Appl. Microbiol. Biotechnol. 62:76-82.2003) have described a new mutant strain of E. coli for pyruvate production. This strain contains three mutations, ppc (phosphoenolpyruvate carboxylase), aceF (pyruvate dehydrogenase), and adhE (alcohol dehydrogenase) and is capable of producing 0.65 grams pyruvate per gram of glucose using complex media supplemented with acetate.
Typical production rates of pyruvate for biocatalysts are around 1 g L−1 h−1 with yields exceeding half the weight of substrate. Torulopsis glabrata, the yeast strain currently used for the commercial production of pyruvate, can achieve pyruvate titers of 69 g L−1. As noted above, T. glabrata strains used in the commercial process are multivitamin auxotrophs requiring tight regulation of vitamin concentrations which result in complex vitamin feeding strategies during fermentation (Li, Y., J. Chen, and S.-Y. Lun, 2001 “Biotechnological production of pyruvic acid” Appl. Microbiol. Biotechnol. 57:451-459). Previous E. coli strains constructed for pyruvate production were cultured in complex media and have been plagued by low titers and yields (Tomar, A. et al. 2003, “The effect of acetate pathway mutations on the production of pyruvate in Escherichia coli.” Appl. Microbiol. Biotechnol. 62:76-82; Yokota A. et al., 1994 “Pyruvic acid production by an F1-ATPase-defective mutant of Escherichia coli W1485lip2.” Biosci. Biotech. Biochem. 58:2164-6167).
Nutrients in culture medium often represent a major cost associated with commercial fermentations. The use of a mineral salts medium and inexpensive carbon source offers the potential to improve the economics of many biological processes by reducing the costs of materials, product purification, and waste disposal (Zhang, J. and R. Greasham, 1999. Appl. Microbiol. Biotechnol. 51:407-421).
There is a need in the art to identify and develop new, efficient, and environmentally friendly processes for producing specialty compounds.